Chemically sensitive organic dye reporters have been developed for over 100 years, and research in this area is still active. However, there are few purely organic sensors that are quantitative, selective, and photochemically stable. Many look to nanotechnology to address these issues. Semiconductor quantum dots (“QDs”, also known as nanocrystals) are highly attractive for making fluorescence measurements due to their extreme brightness and photostability. Unfortunately, the large size and surface passivation of high quality QDs generally prevent chemical or biological sensitivity.
The use of quantum dots for chemical sensing brings significant advantages over organic-based technology alone due to their unique optical and electronic properties. QD color can be tuned through the effect of quantum confinement, meaning that small QDs have much larger bandgaps compared to large QDs. Furthermore, the chemical composition of the QDs may be altered for the purpose of bandgap engineering and to reduce toxicity. As such, utilizing photochemically stable QDs as chemical and biological sensing agents has become a significant research endeavor.
This photochemical stability actually introduces a problem concerning the use of QDs as sensors, as they intrinsically are insensitive to their environment and thus are poor sensors on their own. However, several research groups, including inventors of the present disclosure, have shown that manipulating energy transfer can impart chemical sensing capability to QDs. In 2001, a paper by Van Orden, et al., first demonstrated efficient Förster Resonant Energy Transfer (FRET) from a QD donor to an organic dye acceptor. Van Orden, A., et al., CdSe—ZnS Quantum Dots as Resonance Energy Transfer Donors in a Model Protein-Protein Binding Assay, Nano Lett. 2001, 1, 469-474. Another paper by Mattoussi, et al., showed that QDs can sense chemical agents by designing a CdSe-fluorescence quencher conjugate where the quencher was permanently displaced by trinitrotoluene (TNT). Thus, FRET from the QD to the quencher was removed in the presence of TNT resulting in increased QD emission. Mattoussi, H., et al., A Hybrid Quantum Dot-Antibody Fragment Fluorescence Resonance Energy Transfer-Based TNT Sensor. J. Am. Chem. Soc. 2005, 127, 6744-6751. FRET-based sensing was extended to detect biological species such as maltose. Mauro, J. M. et al., Self-Assembled Nanoscale Biosensors Based on Quantum Dot Fret Donors. Nat. Mater. 2003, 2, 630-638.
There is an ongoing debate concerning the concentration of hydrogen sulfide in cells, blood, and in tissues. A wide range of 2-300 μM has been reported by different groups, which is likely the result of the use of different sampling techniques and detection methods. Concentrations in the nanomolar range have also been reported. Methods of H2S detection such as chromatography, colorimetry, and electrochemical assays suffer from poor biological compatibility, and require complicated sample preparation processes. One strategy to address these issues is based on the fact that H2S dissociates in an aqueous solution to form an equilibrium between H2SHS−S2−, where bisulfide (HS−) is favored and is the target analyte “stand-in” for H2S. As such, the design of fluorescent probes for bisulfide have attracted significant attention due to the convenience, compatibility, and sensitivity of fluorescence methods that facilitate the real-time detection of the analyte within biological environments.
Presently, there are several examples of organic-based sulfide-reactive fluorescent probes that function according to strategies based on metal-sulfide interactions, reduction of azide and nitro groups, and nuclephilic addition. Detailed mechanisms and discussion of these systems can be found in recent reviews, such as Jiang, L. et al., Fluorescence Chemosensors for Hydrogen Sulfide Detection in Biological Systems. Analyst 2015, 140, 1772-1786; and Wang, B. et al., Thiol Reactive Probes and Chemosensors. Sensors (Basel) 2012, 12, 15907-15946. These examples demonstrate sensing organic dyes that brighten in the presence of HS−. However, this single response to the analyte is difficult to calibrate within complex biological environments and may have unknown interactions with other species. This issue is addressed by the use of ratiometrically reporting chromophores, which change color in the presence of the analyte. These systems have an isosbestic point allowing the sensor to be calibrated by measuring the ratio of intensity at any two wavelengths, which is unique for the concentration of the analyte. As a result, the spectrum of the probe provides the analytical metric, rather than the fluorescent probe intensity. While ratiometric or ‘self-calibrating’ fluorescent organic dyes that sense HS− have been reported, the use of these materials may require complex and costly excitation schemes. Furthermore, all organic chromophores are prone towards photobleaching.
A novel ratiometric sensing agent that addresses one or more of the concerns with conventional sensing agents, as discussed above, would be considered a valuable addition to the art.